Alternative molecular mechanisms for force transmission at adherens junctions via β-catenin-vinculin interaction

Force transmission through adherens junctions (AJs) is crucial for multicellular organization, wound healing and tissue regeneration. Recent studies shed light on the molecular mechanisms of mechanotransduction at the AJs. However, the canonical model fails to explain force transmission when essential proteins of the mechanotransduction module are mutated or missing. Here, we demonstrate that, in absence of α-catenin, β-catenin can directly and functionally interact with vinculin in its open conformation, bearing physiological forces. Furthermore, we found that β-catenin can prevent vinculin autoinhibition in the presence of α-catenin by occupying vinculin´s head-tail interaction site, thus preserving force transmission capability. Taken together, our findings suggest a multi-step force transmission process at AJs, where α-catenin and β-catenin can alternatively and cooperatively interact with vinculin. This can explain the graded responses needed to maintain tissue mechanical homeostasis and, importantly, unveils a force-bearing mechanism involving β-catenin and extended vinculin that can potentially explain the underlying process enabling collective invasion of metastatic cells lacking α-catenin.

3OVKRWJK VKH DWVKRTU KDXH HZSDQGHG FRPSWVDVLRQDO DQDO[UHU RI XLQFWOLQ&_&FDVHQLQ LQVHTDFVLRQU LQ the revised manuscript, concerns still remain about the lack of experimental validation to support the predictions from modeling.Authors acknowledge the need for experimental validation of the computational results but defer this to future studies.However, some mutational analysis or binding assays are necessary to back up the modeling, especially for newly proposed interactions.BLVKRWV HZSHTLPHQVDO FRQILTPDVLRQ% VKH STHGLFVLRQU VKDV _&FDVHQLQ FDQ UVLOO ELQG VKH 3.): XLQFWOLQ mutant, or interact with the S2 site, remain speculative.Hence, concerns raised previously have not been adequately addressed here.
4. Reviewer Concern: The authors estimate protein-protein complex stability using NAMD energy profiles rather than the MMPBSA.The latter is better suited for calculating binding affinities and free energy changes in a solvent environment.NAMD calculates the energies of molecular systems by solving the classical equations of motion, whereas MMPBSA (Molecular Mechanics Poisson Boltzmann Surface Area) calculates the free energy of a complex of proteins and/or ligands using molecular mechanics and continuum electrostatics.Further, MMPBSA explicitly models the solvent environment, which can be important for understanding the effect of solvent on the energetics of the system.Additionally, NAMD energy values listed are quite confusing.In some places the energy is written as a positive entity (Fig. 6), though it is negative in the graphs.
Reviewer Comment: While the authors have now emphasized the importance of new coulombic interactions and identified key residues involved through MD studies, in silico mutagenesis studies UKRWOG EH SHTIRTPHG VR XDOLGDVH VKH FRQVTLEWVLRQ RI VKHUH THULGWHU VR VKH UVDELOLV[ RI VKH XLQFWOLQ&_& catenin complex.Furthermore, functional validation studies, such as site-directed mutagenesis/binding analyses are needed to provide experimental evidence to support the computational findings.

Reviewer Concern:
The investigators heavily rely on z-positioning measurements at AJs to UWSSRTV VKH K[SRVKHULU VKDV `&FDVHQLQ HQJDJHU XLQFWOLQ LQ VKH DEUHQFH RI #-catenin.This analysis requires formation of a ROI around the fluorophore where the median position is recorded.Is the difference in z-position affected by the signal intensity?For example, if there is an increase or GHFTHDUH LQ XLQFWOLQ RT `&FDVHQLQ DV VKH PHPETDQH% STRZLPDO VR VKH FDGKHTLQ&FDVHQLQ OD[HT% FRWOG LV DIIHFV VKH \&SRULVLRQLQJ THDGRWV2 @KLU LU LQVHTHUVLQJ FRQULGHTLQJ GLIIHTHQFHU LQ `&FDVHQLQ OHXHOU DV the membrane in the different cell lines (Fig. 4D-E).
Author's Response: Detailed SAIM annotation has now been included in the method section and additional image clarity and explanation provided.
Reviewer Comment: The revisions adequately tackle the technical points raised regarding SAIM data analysis.The clarifications on SAIM principles and added controls support the z-positioning PHDUWTHPHQVU IRT XLQFWOLQ&`&FDVHQLQ ELQGLQJ' @KH DGGHG GHUFTLSVLRQ LQ VKH <HVKRGU STRXLGHU helpful clarification on how SAIM works and provide a satisfactory explanation about SAIM zposition measurements.The authors cite their recent book chapter on SAIM theory to justify how the z-position is calculated from the shape of the measured intensity curve versus incident angle, alleviating concerns about z-position baseline measurements.Image clarification and western blots showing expression levels address the query regarding potential effects of varying protein amounts across cell lines.The authors have effectively responded to the concerns and strengthened the study.
6. Reviewer Concern: As vinculin showed the largest z displacement, what are the vinculin expression levels in these cell lines?This is relevant especially in the context of Figure 3A.If the A50I mutation is used to inhibit localization to focal adhesions, then is an increase in total localization of vinculin at AJs is expected?What percent of total vinculin is going to the AJ?A western blot and corresponding RT-PCR experiment to show differences in vinculin expression in the supplemental materials would be beneficial.
Author's Response: Co-immunoprecipitation data have now been included for the different vinculin mutants to show the expression levels (Sup./' >HXLHYHT 5RQFHTQ1 @KH DWVKRTU^K[SRVKHULU VKDV `&FDVHQLQ HQJDJHU XLQFWOLQ LQ LVU DFVLXDVHG UVDVH IROORYLQJ _&FDVHQLQ GHSOHVLRQ LU EDUHG RQ ILQGLQJU LQ 8LJ +3&5' 3U QRVHG LQ STHXLRWU UVWGLHU% RVKHT vinculin interacting proteins (e.g.Ena/VASP) form distinct and independent mechanical connection with the actin cytoskeleton (Scott, J.A et al. (2005). MBoC, 17 3, 1085-95., Oldenburg, J. et. al. Sci Rep 5, 17225 (2015).https://doi.org/10.1038/srep17225H. Clarity and context: lucidity of abstract/summary, appropriateness of abstract, introduction and conclusions.The abstract concisely summarizes the main findings.The introduction explains the background and goals of the study.The conclusions discuss the impact of the results without over extending interpretation of their findings.Overall, these sections are written clearly and provide suitable framing for understanding the study and its significance.
Reviewer #3 (Remarks to the Author): I am a new reviewer to this revised manuscript by Morales-Camilo.The manuscript focuses on how vinculin, in the absence of alpha-catenin can bind to beta-catenin.The manuscript uses a variety of computational analysis of protein structure, as well as experimental models to increase understanding of how vinculin interacts with catenins at cell-cell adhesions.
Major concerns: 1.In the last response to reviewers the authors mentioned that there were no alpha-catenin KO MDCK cell lines available.This is not correct.MDCK alpha-catenin KO cells using crispr were developed by Ozawa, Biology Open 2018.A more recent publication (Bejar-Padilla, Molecular Biology of the Cell, 2022) developed a similar line.Although the authors show clear differences between WT and KD cell lines, it is not clear if the residual alpha-catenin in the KD cells is impacting things.It would have been much stronger to demonstrate the vinculin to beta-catenin interaction in alpha-catenin KO cells, to demonstrate alpha-catenin is not mediating this interaction.
2. In this revision the authors have added new details about PC3 cancer cells (prostate) suggesting that these cells may be collectively migrating using the beta-catenin vinculin interaction, given low alpha-catenin levels.To me this seems speculative and no efforts were made to modulate these cells to test this hypothesis.3. Does vinculin T12 localize more strongly to cell-cell adhesions?I think this could be an important point to assess, since vinculin must traffic between focal adhesions and cell-cell adhesions, it needs to be made clear if the T12 vinculin to beta-catenin interaction is a result of the T12 mutations directly, or instead indirectly a result of the vinculin being more present at cellcell adhesions.4. Figure 4G: I had a hard time understanding if the differences in migration were significant.Are the migration velocities different?I watched the movies, but was unsure.Also the graphs shown, with regard to direction have no error bars and it is difficult to to know if the differences are meaningful.5.The data presented in Figure 4A (recoil speed) is a different finding than a recent paper (Mezher, Biophys J 2023) that showed no major differences in inter-cellular forces in alpha-catenin KO cells.While these are two different methods (and may be measuring different things) the authors should directly discuss this other manuscript.I note the manuscript is already cited (#35) in the introduction.The Mezher manuscript also cites the pre-print of the present paper mentioning that "only a minor decrease in cell-cell tension as assessed by the retraction of the ablated ends of cell-cell contacts".I am not sure if the authors agree or disagree with the Mezher paper calling the effect "minor"--but I think they should discuss it nonetheless.

Specific Responses to Reviewer's comment:
Reviewer #1 We are glad to see that reviewer 1 is pleased with the large majority of our responses and that they agree that the extensive work done during our first revision has greatly improved the manuscript.Reviewer 1 further requested A) experimental validation through site mutagenesis of the results of MAE;LCAE[T\;9K=EAE DFC=;LC9I DF<=CAE? 9E< #`K@= HL9EKA>A;9KAFE F> MAE;LCAE JG=;A>A; CF;9CAQ9KAFE 9K the cell-cell junction.
Reviewer Comment: While the authors have now emphasized the importance of new coulombic interactions and identified key residues involved through MD studies, in silico mutagenesis studies J@FLC< := G=I>FID=< KF M9CA<9K= K@= ;FEKIA:LKAFE F> K@=J= I=JA<L=J KF K@= JK9:ACAKP F> K@= MAE;LCAE\T\ catenin complex.Furthermore, functional validation studies, such as site-directed mutagenesis/binding analyses are needed to provide experimental evidence to support the computational findings.
Our response, second revision: We entirely agree with the reviewer that in silico and in vitro site mutagenesis and biochemical confirmation would further validate the MD analysis to confirm the predicted sites of interactions >FI K@= MAE;LCAE[T\;9K=EAE <AD=IY *E K@= GI=J=EK M=IJAFEW N= <AJ;LJJ=< K@= =OG=IAD=EK9C M9CA<9KAFE F> K@= AEK=I9;KAFE F> T\;9K=EAE 5#3 NAK@ 3g _5%g9`9E< 3h _5%g:`JAK=JW N@A;@ N=I= GI=MAFLJCP published by co-authors of the current manuscript (Le et al. Sci Adv. 2019).While the cited work does not elucidate the exact binding sites, it supports the general molecular arrangement that our MD analysis hints to.Furthermore, we would kindly point out that the site mutagenesis of each of the residues predicted by MD would require a huge investment of time and resources (also considering that the work is conducted in a resource-limited environment) to validate a point that is only partially relevant for the present work.As indicated by the new title, which as a matter of fact was inspired by the reviewer´s comment regarding the novelty of our work, our manuscript focuses on the newly <AJ;FM=I=< MAE;LCAE[U\;9K=EAE >LE;KAFE9C AEK=I9;KAFEJY *DGFIK9EKCPW K@= I=MA=N=I J==DJ KF := >LCCP satisfied by the extensive body of evidence provided (super-resolution microscopy, FRAP, laser ablation, co-immunoprecipitation) and now this key point is further strengthened by additional single molecule magnetic tweezer experiments.We also wish to stress the fact that, in this work, we used MD analysis as a power comparative tool to make prediction of the relative importance (based on energetic considerations) and possible configurations that allowed to propose the functional model reported in figure 6.Nevertheless, we fully agree with the reviewer that our in-silico data alone are not sufficient to make JKIFE?JK9K=D=EKJ I=?9I<AE?K@= JG=;A>A; I=JA<L=J D=<A9KAE?T\;9K=EAE\MAE;LCAE AEK=I9;KAFEY "CJFW residues reported for the other interactions would require similar scrutiny and further experimental validation, which would account for a tremendous amount of work, which we can only defer to a future publication.Thus, we further emphasize throughout the manuscript that experimental evidence is needed to validate the MD analysis and we trust that reviewer will consider this as an agreeable compromise, also in view of the fact that the manuscript has been transferred to an interdisciplinary journal, where a large portion of the audience may not be as interested in the molecular details.

Our response second revision:
As suggested by the reviewer we have analyzed relative distribution of vinculin at the cell-cell junction versus the total vinculin.The results of this analysis are shown in Supplementary Figure 5B and C and support the lower recruitment of vinculin T12-A50I mutant to cadherin-mediated adhesions respect to Vinculin wt and T12 mutant.This has been further validated using a newly ?=E=I9K=<T\;9K=EAE ,0 ;=CC CAE=Y 6= K@9EB K@= I=MA=N=I >FI K@AJ JL??=JKAFE 9J K@= HL9EKA>A;9KAFE allowed us to validate the results obtained by MD (Figure 3C-D), co-IP (Suppl.Figure 10), SAIM (Figure 3A) and the functional analysis shown in Figure 4C to J.
The main text reports this analysis at lines 215-217: ¨This [vinculin localization] N9J M=IA>A=< AE :FK@ T\;9K=EAE BEF;B<FNE 9J N=CC 9J BEF;BFLK ;FE<AKAFEJ confirming that the recruitment of active vinculin to the cadherin-catenin layer is not due to the DAEAD9C 9DFLEK F> C=>KFM=I T\;9K=EAE GI=J=EK AE K@= .%$,T\;9K=EAE ,% _3LGGC=D=EK9IP 'A?LI= k and 6).¨And at lines 401-404: R*DGFIK9EKCPW =HL9C 9DFLEKJ F> MAE;LCAE NK 9E< MAE;LCAE 4gh AE .%$,NK 9E< .%$,T\;9K=EAE ,% were measured at the AJ (Supplementary Figure 5B and C), thus proving that results from laser ablation, FRAP and wound-model assay should be interpreted as consequence of the type of vinculin configuration/mutation and not to the relative amount of protein at the AJ.¨ Reviewer #3: We are grateful to the reviewer for stepping in and providing us with their valuable feedback and expert comments.Much appreciated!Major concerns: 1.In the last response to reviewers the authors mentioned that there were no alpha-catenin KO MDCK cell lines available.This is not correct.MDCK alpha-catenin KO cells using crispr were developed by Ozawa, Biology Open 2018.A more recent publication (Bejar-Padilla, Molecular Biology of the Cell, 2022) developed a similar line.Although the authors show clear differences between WT and KD cell lines, it is not clear if the residual alpha-catenin in the KD cells is impacting things.It would have been much stronger to demonstrate the vinculin to beta-catenin interaction in alpha-catenin KO cells, to demonstrate alpha-catenin is not mediating this interaction.
2. In this revision the authors have added new details about PC3 cancer cells (prostate) suggesting that these cells may be collectively migrating using the beta-catenin vinculin interaction, given low alpha-catenin levels.To me this seems speculative and no efforts were made to modulate these cells to test this hypothesis.
The reviewer is certainly correct to point out that this observation is speculative and demonstration of pathological relevance of our investigation would still requires further in-depth investigation.However, we wish to reassure the reviewer that we did not base our statement on a wild guess/assumption.A main effort of our laboratory is now directed to demonstrate the pathological I=C=M9E;= F> U\;9K=EAE[MAE;LCAE AE GIFJK9K= ;9E;=I ;=CC CAE=JY 1I=CADAE9IP <9K9 J@FN K@9KW 9J =OG=;K=<W cell-cell junctions in PC3 are not as stable as those of normal epithelial cells (due to the absence of T\;9K=EAE`Y $FEJ=HL=EKCPW 9 JD9CC <=?I== F> JAE?C=\;=CC J;9KK=IAE?9CFE?NAK@ ;=CC JKI=9DAE?AJ JKACC <=K=;K9:C=Y *EK=I=JKAE?CPW I=J;L= =OG=IAD=EKJ N@=I= T\;9K=EAE AJ BEF;B\AE AE 1$i ;=CCJ J@FN 9 transition to strong collective migration and complete inhibition of residual single cell migration, as shown in the figure below. As we agree with the reviewer, we have modified the manuscript to clearly indicate that this point remains speculative at this time and further analysis would be needed.However, we do not feel it would be appropriate to entirely remove it from the manuscript as the supplementary video 11 was specifically requested and accepted by reviewer 1 who asked to suggest possible physiological or pathological scenarios relevant to our findings.In principle, we could also agree to remove entirely this point from the manuscript (also because we are working on expanding this interesting point in future publications).Thus, we brought it to the attention of the editor who we believe should also arbitrate eventual divergence of opinion between the reviewers.To avoid misleading the readers, we clarified as honestly as we could how to interpret those data.
The main text has been modified as follows at line 404-408: ¨For instance, such novel function could potentially explain how PC3 cells, which are derived from GIFJK9K= ;9E;=IW 9I= ;9G9:C= F> DA?I9KAE?;FCC=;KAM=CP _3LGGC=D=EK9IP MA<=F gg`<=JGAK= C9;BAE?T\ catenin (expression levels in Supplementary Figure 4C-D).However, this observation remains only JL??=JKAM= F> GFJJA:C= ;FE<AKAFEJ N@=I= >FI;= KI9EJDAJJAFE :P K@= U\;9K=EAE[MAE;LCAE ;FDGC=O ;9E possibly explain yet unresolved pathological scenarios.¨3. Does vinculin T12 localize more strongly to cell-cell adhesions?I think this could be an important point to assess, since vinculin must traffic between focal adhesions and cell-cell adhesions, it needs to be made clear if the T12 vinculin to beta-catenin interaction is a result of the T12 mutations directly, or instead indirectly a result of the vinculin being more present at cell-cell adhesions.
We thank the reviewer for raising this important point.We quantified the relative distribution of exogenous vinculin at the AJ vs cytosolic levels (indicative of protein recruitment) at cell-cell junction AE .%$,NK 9E< .%$,T\;9K=EAE ,% KI9EJ>=;K=< NAK@ MAE;LCAE NKW 4gh 9E< 4gh\"kf*Y 0ECP 4gh\"kf* showed a lower level of recruitment at the AJ (Supplementary figure 5B).The results of vinculin T12 N9J 9CJF ;FIIF:FI9K=< LJAE? .%$,T\;9K=EAE ,0 ;=CCJ _3LGGC=D=EK9IP >A?LI= l$W &W %`Y 4@=J= I=JLCKJ prove that the functional studies here reported are to be attributed to the type of vinculin configuration/mutation rather than its amount at the AJ.This has been included in the main text at lines 401-404 that now read: R*DGFIK9EKCPW =HL9C 9DFLEKJ F> MAE;LCAE NK 9E< MAE;LCAE 4gh AE .%$,NK 9E< .%$,T\;9K=EAE ,% were measured at the AJ (Supplementary Figure 5B), thus proving that results from laser ablation, FRAP and wound-model assay should be interpreted as consequence of the type of vinculin configuration/mutation and not to the relative amount of protein at the AJ.¨ 4. Figure 4G: I had a hard time understanding if the differences in migration were significant.Are the migration velocities different?I watched the movies, but was unsure.Also the graphs shown, with regard to direction have no error bars and it is difficult to to know if the differences are meaningful.
We apologize for the poor representation of our wound-model assays.Following the reviewer's advice, we have largely modified this session.Most importantly, we have: 1) Included the standard deviation to the relevant figure panels (now Figure 4I and J); 2) Provided the relative angular speed (which describes the difference in tissue motion for each angular sector) in addition to the mare frequency distribution; 3) *E;CL<=< JK9KAJKA;9C 9E9CPJAJ K@9K <=DFEJKI9K=J K@9K .%$,NK 9E< .%$,T\;9K=EAE ,% NAK@ MAE;LCAE 4gh 9I= JK9KAJKA;9CCP M=IP JADAC9IW N@=I=9J .%$,T\;9K=EAE ,% :=@9M= C=JJ collectively; 4) Provided the quiver representations of the PIV analysis in the supplementary movies.We believe that this last quite clearly shows loss of collective migration (laminar flow) in MDCK T\;9K=EAE ,% 9J ;FDG9I=< KF K@= FK@=I ;FE<AKAFEJW N@A;@ CFFB HLAK= JADAC9I KF =9;@ FK@=IY These changes can be found various parts of the main text, figures and supporting material: -Figure 4I and J now substitute Figure 4G; -New Supplementary Movies containing the PIV quiver have been uploaded (Supplementary Video 8A, B and C); -Supplementary Table 4 reports the statistical analysis; -Experimental method, PIV analysis and statistics have been expanded in the methods.
The relevant paragraph in the main text at lines 380-401 now reads: ¨Finally, to further test force transmission between cells under these conditions and evaluate their large-scale consequences, we probed for the ability of cells to maintain collective migration in a wound-model assay (Figure 4I-J, Supplementary table 4 and Supplementary Video 8A-C).Particle Image Velocimetry (PIV) analysis of these experiments, which vectorially represents the flow of cells, showed that expectedly MDCK wt cells migrate cohesively by coordinated collective migration (Supplementary Video 8A) and the preponderant frequency distribution (Figure 4I, left) as well as the relative angular speed (Figure 4J, left) being perpendicular to the migrating front (i.e., 0°).In ;FEKI9JKW ;@9FKA; DFKACAKP 9E< <=;I=9J=< ;FFI<AE9KAFE N9J F:J=IM=< AE .%$,T\;9K=EAE ,% 9J consequence of partial loss of force transmission between cells (Supplementary Video 8A) as indicated by the lower frequency distribution and relative angular speed perpendicular to the front of the tissue edge (i.e., 0°) (Figure 4I and J, middle panels).Interestingly, Supplementary Video 8C 9E< IA?@K G9E=CJ AE 'A?LI= j* 9E< + <=DFEJKI9K= K@9K =OGI=JJAFE F> 4gh AE T\;9K=EAE ,% ;=CCJ N9J 9:C= to largely rescue the cohesive migration phenotype seen in wildtype cells.It must be noted that by experimental design, contact inhibited cells, such as MDCK cells, will be prevented from migrating toward the bulk of the tissue and thus a preferential motion perpendicular to edge of the tissue (along the x axis) is to be expected.As result, motion is directionally skewed, and statistical analysis has reduced degrees of freedom.Nonetheless, statistical analysis indicates high correspondence :=KN==E DF<= F> DA?I9KAFE F> .%$,NK 9E< .%$,T\;9K=EAE ,% KI9EJ>=;K=< NAK@ MAE;LCAE 4gh mutant (p values = 0.81 and 0.97 for the angular frequency and mean speed magnitude, respectively ] 3LGGC=D=EK9IP 49:C= j`Y $FE;FDAK9EKCPW CFN JADAC9IAKP N9J F:J=IM=< N@=E ;FDG9IAE?.%$,T\ ;9K=EAE ,% NAK@ K@= FK@=I J9DGC=J _fYgm r G M9CL=J r fYim >FI 9CC J9DGC=J N@=E ;FDG9I=< KF T\ catenin KD).¨ 5.The data presented in Figure 4A (recoil speed) is a different finding than a recent paper (Mezher, Biophys J 2023) that showed no major differences in inter-cellular forces in alpha-catenin KO cells.While these are two different methods (and may be measuring different things) the authors should directly discuss this other manuscript.I note the manuscript is already cited (#35) in the introduction.The Mezher manuscript also cites the pre-print of the present paper mentioning that "only a minor decrease in cell-cell tension as assessed by the retraction of the ablated ends of cellcell contacts".I am not sure if the authors agree or disagree with the Mezher paper calling the effect "minor"--but I think they should discuss it nonetheless.*E 9 ELKJ@=CCW N= 9?I== NAK@ K@= 9LK@FIJ K@9K 9CK=IE9KAM= G9K@J :PG9JJAE?T\;9K=EAE ;9E D=<A9K= >FI;= KI9EJDAJJAFE 9K K@= "+Y )FN=M=IW N= <AJ9?I== NAK@ K@=AI MA=N K@9K ;FEJ=HL=EKCP T\;9K=EAE AJ not important.
It has to be noted that indeed laser ablation measures elastic tension built along the cell-cell junctions whereas the method described by Mezher et al., which is derived from the group of Margaret Gardel (Maruthamuthu et al. PNAS 2011), measures normal forces perpendicular to the AJ.Another main difference is that the forces we measure are in confluent epithelial tissues, whereas they perform their measurements in cell aggregates composed by 2-3 cells, where the junction is likely to be less mature as compared to full confluency.Despite these differences, we do not understand what justified the authors assessment that our C9J=I 9:C9KAFE =OG=IAD=EKJ J@FN RFECP DAEFIR =>>=;KJY *E 9:J=E;= F> T\;9K=EAE N= J== I=C=M9EK 9E< significant reduction in tension.Similarly wound model assays are also in agreement with this loss of function.Furthermore, several groups, including our, consistently reported this effect over the years and in several publications (e.g., Ravasio et at. Integrative Biol. 2015, Seddeki et al. MboC 2018, Barry KA et al J Cell Sci. 2014, etc).At last, the same authors seem to have contradictory results as figure 5 of the same paper show K@9K T\;9K=EAE AJ ;IL;A9C KF D9AEK9AE ;F@=JAM=E=JJ F> K@= KAJJL= AE :A9OA9C JKI=K;@AE?=OG=IAD=EKJY We have explicitly argued about this point in the discussion of the paper that now at lines 451-456 reads: ¨While this view a;=EKI9C ADGFIK9E;= F> T\;9K=EAE AE >FI;= KI9EJDAJJAFE 9K K@= "+b has been recently challenged 35 , our results, based on direct measurement of tensional state of AJ, clearly demonstrate a significant loss F> K=EJAFE AE T\;9K=EAE ,% ;=CCJ _'A?LI= j%\&`W ;FE>AIDAE?K@= ;=EKI9C IFC= F> T\;9K=EAE LE<=I physiological conditions.However, growing body of evidence suggests that alternative pathways could mediate force transmission at the AJ 29-35 Y )=I=W N= <=DFEJKI9K= K@9K U\;9K=EAE ;9E >FID 9 G@PJA;9C ;FEE=;KAFE NAK@ FG=E MAE;LCAEW ;9G9:C= F> I=J;LAE? >FI;= KI9EJDAJJAFE AE 9:J=E;= F> T\ catenin.¨I think the authors have done an excellent job of addressing my and the other reviewer's comments.The revised manuscript clearly demonstrates there is a vinculin-beta catenin interaction and that this interaction is capable of mechanical force transmission.This is impactful because alphacatenin has been assumed to be the only linker for connecting vinculin to adherens junctions.Regarding the previous concern about vinculin expression levels in different cell lines and their potential impact on the observed z-displacement, the authors have adequately addressed this by analyzing the relative distribution of vinculin at the cell-cell junctions versus the total vinculin.By analyzing the relative distribution of vinculin at the cell-cell junctions versus the total vinculin, they have demonstrated that the lower recruitment of the vinculin T12-A50I mutant to cadherinmediated adhesions compared to wild-type vinculin and the T12 mutant is not due to differences in expression levels.
While the lack of experimental validation of the molecular dynamics simulations for the specific 4*5.)7*50*).&6.1, ;#(&6*1.1#8.1(7/.1 .16*4&(6.2154*0&.15& /.0.6&6.21"6-* &76-245 -&8* acknowledged this and emphasized the need for future experimental validation.Considering the extensive experimental evidence supporting the main conclusions of the study, I believe that this limitation does not detract from the overall impact of the work. Fig 9) and vinculin expression at cell-cell adhesions analyzed via image analysis (Sup Fig 5).Reviewer Comment: Although differences in exogenous vinculin expression levels are shown (Sup Fig 4C), the author's demonstrate vinculin localization to cell-cell adhesions via co-IP (Sup Fig 9) and image analysis (Sup Fig 5).It has been demonstrated that the T12 A50I vinculin mutant does not strongly associate with cell-cell adhesions in the presence of #-catenin KD as seen in the main text.It would be helpful to determine the percentage of vinculin localizing to cell-cell adhesions with respect to total vinculin localization.
Author's Response: Co-immunoprecipitation data have now been included for the different vinculin mutants to show the expression levels (Sup.Fig 9) and vinculin expression at cell-cell adhesions analyzed via image analysis (Sup Fig 5).Reviewer Comment: Although differences in exogenous vinculin expression levels are shown (Sup Fig 4C), the author's demonstrate vinculin localization to cell-cell adhesions via co-IP (Sup Fig 9) and image analysis (Sup Fig 5).It has been demonstrated that the T12 A50I vinculin mutant does not strongly associate with cell-cell adhesions in the presence of #-catenin KD as seen in the main text.